This invention relates to a detector system for performing sample analysis, such as DNA sequencing, DNA fingerprinting, gene expression analysis, and the like. More particularly, it pertains to a detector system which employs a transmission grating beam splitter for separating incoming light, either fluoresced or otherwise emitted from samples separated from one another in a two-dimensional array, into multiple order diffraction bands and wavelengths.
Genetic sequence information, such as that obtained in the Human Genome Project, has applications in functional genomic research, disease diagnostics, drug discovery, and the like. Micro-array technologies have arisen to meet the demand for expanding the acquisition and utilization of the genetic information. These technologies often involve imaging a microfabricated array of probe sequences disposed on a support, such as a microchip, slide, array or chip. For example, genetic mutations may be detected by a method known as sequencing by hybridization. In sequencing by hybridization, a solution containing one or more targets to be sequenced (i.e., samples from patients) contacts multiple DNA probes arranged in a microfabricated array. The targets will bind or hybridize with probes in the array that contain complementary sequences. Generally, the targets are labeled with a fluorescent marker, radioactive isotopes, enzymes, or other types of markers. Accordingly, locations at which targets hybridize with complimentary probes can be identified by locating the markers. Based on the locations where hybridization occur, information regarding the target sequences can be extracted. The existence of a mutation may be determined by comparing the target sequence with the wild type.
Microchip fabrication technology has recently entered the commercialization stage in which multiple vendors provide robotic microchip printing devices for users to customize their own chips for specific studies, including gene expression, genotyping, sequencing, and the like. The printing devices generate two-dimensional arrays of spots or analysis sites. A site refers to an area or region of a substrate that accommodates a fluorophore or is functionalized to provide a response indicative of the amount of a target substance present in a sample. Typically, different sites are functionalized to provide a response indicative of different targets. To improve precision, however, more than one site can be functionalized to respond to the same target. Preferably, the sites are arranged as an array of discrete spots, with each spot being hybridized with one or more different probes.
Stand-alone array printers can be purchased separately for less than about $5000. These printing devices can automatically print hundreds of chips, or slides, per load. The typical spot size printed on a chip is about 20-100 xcexcm. Typically, the spots are printed with strands of DNA, antibodies, or the like, configured to indicate the presence of or amount of a particular target present in a sample. Spot sizes of tens of microns or more generate relatively low density microchips. Low-density microchips, however, are more practical when researchers have identified a limited set of targets, such as a specific groups of genes, they wish to study or use as diagnostic markers. More difficult diagnostic and research assays, in contrast, require higher density arrays at least in part because less information is known about the sample prior to the analysis. Higher density arrays of the type required for these analyses are produced by, for example, Affymetrix using lithographic techniques. In some cases, feature sites may be as small as a few microns or even a single molecule. For example, about 105 to 106 features may be fabricated in an area of only 12.8 mm2.
Because of their smaller spot size and more compact architecture, the high density arrays require high resolution optical devices or readers to interrogate the sites of the arrays. Several vendors have manufactured microchip reading devices to detect the signals from these chips. Currently, all these devices are based on fluorescence detection, which is the detection of fluoresced light resulting from one or more fluorophores upon exposure of the fluorophores to an excitation light. They employ either confocal scanners combined with photomultiplier tube (PMT) detectors (for companies such as Genetic MicroSystems, GSI Lumonics, Virtek Vision), or a CCD detector with an imaging lens (Hitachi). These devices can detect two dyes, such as Cy3 and CyS, by alternating the excitation laser wavelength to match a specific dye excitation spectral maximum, and/or switching an optical filter to match the fluorescent spectrum of a particular dye. Neither of these devices has a spectrometer capable of simultaneous multi-wavelength detection to simultaneously distinguish multiple dyes from sites within an array. Additionally, confocal scanners require precisely controlled movement of either the scanner head or the chip in three dimensions, which compromise the robustness of the instrument.
Determining the presence of multiple fluorophores within an array has biologically important applications. For example, the probe arrays may be designed specifically to detect genetic diseases, either from acquired or inherited mutations in an individual DNA. These include genetic diseases such as cystic fibrosis, diabetes, and muscular dystrophy, as well as acquired diseases such as cancer. Microchip arrays have proved to be a high-throughput method for gene expression studies, capable of screening thousands of genes on a single microchip. In order to extract the meaningful information from such large amount of gene expression data from one biological sample, one has to compare it to a reference sample in order to obtain the differential expression pattern. The reproducibility of the response obtained from individual sites in an array of sites is influenced by a number of experimental factors, such as temperature, the hybridization buffer, the quality of the substrate, the ability to print arrays with precise amounts of material within each site and the like. One way to eliminate uncertainties introduced by these site-to-site variations is to simultaneously measure the response of both the sample and the control for each site. Measuring both a sample and control response from each site, however, requires that the fluorescent detection system be able to distinguish the sample and control responses rapidly and efficiently.
Another demanding example that would require the ability to distinguish multiple responses is the use of microfabricated arrays to monitor changes in gene expression patterns of a cell line during different phases of a drug treatment study. It would be desirable to monitor the gene expression at many points in time, such as five or more times, during the study. Because these gene expression changes can be subtle, a highly sensitive and precise monitoring method is required. If the samples obtained from different phases of the drug treatment are analyzed within different sites in an array, however, the above-mentioned variations in the site to site response can decrease the precision of the measured response, which decreases the value of the information to the researcher or clinician. In order to reduce the experimental variation, the samples from different points during the study would desirably be pooled together and analyzed within the same sites on the array. In general, if N samples were pooled together in a particular site, the number of fluorophores in the site would range from zero to N depending on how many of the samples contain a target having an affinity for a probe disposed at that site. Therefore, the multiplexed sample approach requires a detection system able to distinguish simultaneously the response from as many as N different fluorophores to maximize the sample analysis rate (throughput).
Systems that utilize interchangeable filters or different excitation wavelengths to sequentially measure the response of different fluorophores, have reduced throughput because they cannot simultaneously measure the response from multiple dyes. Moreover, the source light used to excite the fluorescence may photobleach fluorophores in the sample. Therefore, the response of a fluorophore that is measured later in the measuring sequence may be biased lower by the photobleaching.
Therefore, there is a need to simultaneously measure the response of multiple fluorophores from each site within an array. The present invention involves using a spectrometer to spectrally obtain simultaneously the total fluorescence spectrum resulting from multiple fluorophores. Based on the total observed fluorescence spectrum, deconvolution techniques can be used to resolve the amount of multiple individual fluorophores in the sample. Within a given spectral range, deconvolution techniques allow more fluorophores to be resolved than by using filters. Therefore, more samples can be multiplexed within each site of the array to increase throughput and accuracy of differential gene expression measurements. Multiplexing a group of spectrally close dyes allows more efficient excitation using a single excitation source. Wavelength selection using a transmission grating eliminates the need for switching filters, simplifies instrumental design, and increases fluorescence light collection efficiency. The present invention offers significant advantages over the method of using different wavelength lasers to selectively excite each individual dye coupled with narrow band pass filters for selective detection of the dye.
An embodiment of the present invention relates to an array reader comprising a light source configured to emit an excitation light and a substrate having a plurality of discrete sites arranged in at least two dimensions. Each site is configured to support a sample. The array reader includes a detector comprising an array of light sensitive elements and a diffracting element disposed along an optical path between the substrate and the detector. The diffracting element is configured to receive non-collimated light emitted by at least one sample illuminated by said excitation light.
Each site may have associated therewith one or more sequences of nucleotides, proteins or peptides serving as probes. The probes may be integrally bound to substrate.
The substrate can comprise a platform, a microchip, slide, or a microtitre tray having a plurality of wells configured to accommodate samples.
The diffracting element is preferably a transmission grating beam splitter (TGBS). The TGBS separates the non-collimated light received from the at least one sample into a 0th-order component which is received by a first set of said light sensitive elements and a higher-order component which is received by a second set of said light sensitive elements, the second set being spaced apart from the first set. Each member of the second set is disposed at a distance from the first set which is indicative of a wavelength of light received by that member.
In another embodiment, the non-collimated light is received by the TGBS without first having been optically altered by a light focusing element. A single light focusing element is disposed along an optical path segment between the TGBS and the detector.
In yet another embodiment, the array reader comprises a single light focusing element disposed along the optical path between the substrate and the detector, said light focusing element being disposed between the substrate and the TGBS.
The substrate preferably comprises a two-dimensional array of sites arranged as a plurality of rows and a plurality of columns. In one embodiment, the array reader comprises a changing device configured to determine which of said plurality of sites is illuminated at any given instant. The changing device can be configured to alter a position of the sites with respect to the detector. Alternatively, or in combination with this embodiment, the changing device is configured to alter a direction of the excitation light so as to selectively illuminate a subset of said plurality of sites.
Yet another embodiment of the present invention relates to an array reader comprising a light source configured to emit an excitation light, a substrate comprising a plurality of sites spatially configured as a two-dimensional array having a plurality of rows and a plurality of columns, each site configured to support a sample, a changing device configured to determine which of said plurality of sites is illuminated at any given instant, a detector comprising a two-dimensional array of light sensitive elements, a transmission grating beam splitter(TGBS)disposed along an optical path between the substrate and the detector, a single light focusing element disposed along an optical path between the substrate and the detector, wherein the TGBS is configured to receive non-collimated light emitted by at least one sample illuminated by said excitation light, the non-collimated light being received by the TGBS without first having been optically altered by a light focusing element.